Fermentative Hydrogen Production by a Newly Isolated Mesophilic Bacterium HN001 Hiroki NISHIYAMA, Shigeharu TANISHO b Graduate School of Environment and Information Sciences, Yokohama National University Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan b tanisho@ynu.ac.jp ABSTRACT: Fermentative hydrogen at high temperature has been tested using a newly isolated mesophilic bacterium strain HN001. Experimental temperatures and phs were set at 41, 44, 47, 50C and 5.5, 6.0, 6.5, 7.0, respectively. The rate of H 2 from glucose showed the maximum at 47C and ph6.0.the rate was approximately 150 mmol L-culture -1 h -1. The yield of hydrogen from glucose increased along with the increase of temperature and the decrease of ph. The maximum yield of 2.4 mol-h 2 mol-glucose -1 was obtained at 50C and ph5.5. This bacterium grew fast as cultivation temperature becomes high. The of acetate and ethanol increased as the cultivation temperature increase while the of lactate and butyrate decreased. The of butyrate and lactate decreased also along with the increase of ph while the concentration of formate increased KEYWORDS : Hydrogen, Fermentation, Mesophilic bacterium HN001, Cultivation temperature, ph Introduction: Fossil fuels will be exhausted in near future and as a consequence, environmental problems become very serious. As an ultimate clean energy, hydrogen has been expected all over the world. Hydrogen fermentation is very promising H 2 method from wet biomass such as food waste or molasses. But the rate of H 2 was not fast enough for practical use. Recently, hydrogen fermentation at high temperature has been researched for the purpose of accelerating the rate of H 2. Moreover high H 2 yields were acomplished by some of thermophilc bacteria [1]. However, the rates of H 2 of thermophilic bacteria were less rapid than regular bacteria. At the same time, keeping fermenter at high temperature such as 70 C requires large amount of energy. Accordingly, screening of new mesophilic bacteria was carried out. As the results, a new ideal mesophilic bacterium was obtained from sewage. This paper reports properties of this bacterium in hydrogen from glucose such as effects of cultural temperature and ph. Materials and methods: Micro Organism and Culture medium: A mesophilic bacterium newly selected from sewage was used in this experiment. This bacterium is called mesophilic bacterium HN001 in our laboratory. Synthetic culture medium named YNU anaerobic culture was used for the pre- and main cultivation. The nutrient composition of the culture was as follows: casein peptone 25.0g, dried yeast extract-s (Nihonseiyakukougyo) 22.0g, L-cystein hydrochloride monohydrate 0.3g, mercaptoacetic acid 0.3g and glucose 15.0g per 1L liquid. This culture liquid was used without sterilization. Cultivation and Analysis: The mesophilic bacterium HN001 was pre-cultivated in a 16mL ABCM semisolid agar culture (Eiken Chemical) for 16 hours at 20 C and kept at 50 C for 30 min before the inoculation on the main culture to acclimatize to high temperature. Batch cultivation of the mesophilic bacterium HN001 was carried out with a 500 ml jar fermenter. The volume of culture liquid was 350 ml and the liquid stirred by stirrer at 30 rpm. To investigate the effect of cultivation temperature and ph on the hydrogen evolution, temperatures and phs were set at 41, 44, 47, 50 C and 5.5, 6.0, 6.5, 7.0, respectively. The ph was monitored by an automatic ph controller and adjusted to the setted up ph by 1M NaOH liquid. The accumulated volume of H 2 was measured by the liquid displacement method using 1M NaOH liquid to remove CO 2 from product gas. The maximum rate of H 2 and the yield of H 2 were calculated by using the modified Gompertz equation 1/1
[2]. The metabolic products such as acetate, ethanol and lactate were analyzed by a liquid chromatograph (column: GL-C610H-S Hitachi Chemical). Results and Discussion: Effect of cultivation temperature: Figure 1 shows the effect of cultivation temperature on the maximum rate of H 2 making ph as parameter. The maximum rate was determined from one of the parameters of the modified Gompertz equation that is known as a growth curve. All data except at temperature 44 C laid on lines taking ph for parameter. The lines showed convex curve to the cultivation temperature having a peak at 47 C. By this, the optimum temperature for hydrogen was found to be at around 47 C for the strain HN001. The largest rate at the peak was ca. 150 mmol-h 2 L-culture -1 h -1 at ph6.0. This rate is the biggest among the known data listed in a paper [1]. From Fig.1, the optimum ph was also determined. The data on ph6.5 were a little lower than ph6.0 and the data on ph5.5 were lower than ph6.5. From these results, the optimum ph was found to be 6.0. Fermentation products of this bacterium were mainly H 2, CO 2, acetate, ethanol, butyrate, lactate and formate. Figure 2 shows the effect of cultivation temperature on acetate. The concentration of acetate increased as the temperature increase. The trend was clearly seen at the temperature over 45 C. This phenomenon is very important for the biological hydrogen, because many bacteria produce hydrogen by the NADH pathway [3] and surplus NADH is generated through acetate. NADH + H + NAD + + H 2 C 6 H 12 O 6 + 2NAD + 2CH 3 COCOOH + 2NADH CH 3 COCOOH + NAD + + H 2 O CH 3 COOH + CO 2 + NADH + H + Therefore, more H 2 can be obtained when bacteria produce more acetate. Fig.1. The effect of temperature and ph on the maximum H 2 rate Fig.2. The effect of temperature on acetate Figure 3 shows the effect of temperature on ethanol. Ethanol concentration also increased along with the cultivation temperature increase and the curve showed similar trend with the acetate. Additionally, acetate and ethanol concentration became nearly equal at 50 C. Production of ethanol from pyruvate requires 1 mole of NADH as the following reaction. CH 3 COCOOH + NADH + H + CH 3 CH 2 OH + CO 2 + NAD + So when bacterium produces ethanol, yield of hydrogen decreases in contrast. However ethanol is one of the good energy resources. Therefore, if it is separated from fermenter under reduced pressure, it may be used for energy resource. Figure 4 shows the effect of cultivation temperature on butyrate. The butyrate concentration was lower than the other main products as ca. 5 mm at 47C while acetate and ethanol were high as ca. 60mM. It decreased along with the increase of temperature. The of butyrate requires 2 moles of pyruvate. The reaction produces 2 moles of NADH but utilizes 2 moles of NADH simultaneously. After all, butyrate consumes 2 moles of pyruvate without making surplus NADH like the following reaction. 2 CH 3 COCOOH CH 3 CH 2 CH 2 COOH + 2CO 2 2/2
WHEC 16 / 13-16 June 2006 Lyon France Figure 5 shows the effect of temperature on lactate. Although there are wide spread data at 47C, the concentration of lactate decreased along with the increase of temperature. This trend is similar to the butyrate. The pathway of lactate requires 1 mole of NADH as follows, therefore when bacteria produce lactate, the yield of hydrogen decreases theoretically. CH 3 COCOOH + NADH + H + CH 3 CHOHCOOH + NAD + Considering the above reaction, the decrease of lactate at high temperature is a great advantage for hydrogen. C Fig.3. The effect of temperature on ethanol Effect of ph on : Figure 6 shows the effect of ph on the yield of hydrogen. The yield increased along with the decrease of ph. It steeply increased from ph 7 to over around ph 6. This might be resulted from formate decomposition. Since formate is known to be decomposed to H 2 and CO 2 at low ph [3]. Additionally, there was a trend of increasing yield in the increasing temperature. This is resulted from the lactate decrease and acetate increase at high temperature. The effect of ph on acetate was checked, but the correlation was not found between them. The same check was tried on the ethanol but the correlation was not found also. Fig.5. The effect of temperature on lactate Fig.4. The effect of temperature on butyrate 3/3
Figure 7 shows the effect of ph on butyrate. The butyrate concentration decreased along with the increase of ph and it was constant over ph6.5. Although butyrate concentration was totally low, it is revealed that both higher ph and higher temperature repress butyrate Fig.6. The effect of ph on H 2 yield. Fig.7. The effect of ph on butyrate Fig.8 shows the effect of ph on lactate. Although there is a fluctuation, the concentration of lactate decreased along with the increase of ph. From Fig.8, lactate was repressed from high ph and high temperature. The data except 44C showed the clear its trend. So it is assumed that the data on 44C also show the trend like straight line in Fig.8. To keep down the butyrate and lactate s, higher ph (around ph7.0) and higher temperature are available but if ph is set at high, the H 2 yield becomes lower from Fig.6. Thereat high temperature (around 50C ) is enough to repress the butyrate and lactate s and the maximum H 2 rate is fast (Fig.1, Fig.4 and Fig.5). From these results, the optimum conditions about H 2 by Mesophilic bacterium HN001 seem to be lower ph and higher temperature. At the same time, these results are very interesting because the reaction of NAD + /NADH is affected by ph [3]. The redox reaction of NAD + /NADH is concerned in acetate, ethanol, butyrate and lactate s. Therefore the s of acetate, ethanol, butyrate and lactate were expected to be affected by ph. But in this study, acetate and ethanol were not limited by ph. Fig.9 shows the effect of ph on the formate. The concentration of formate drastically decreased linearly along with the decrease of ph. From the previous study, it is revealed that formate is decomposed to H 2 and CO 2 at low ph (about under ph6.4)[3]. This is one of the reasons that higher H 2 Fig.8. The effect of ph on lactate. Fig.9. The effect of ph on formate. 4/4
Conclusions: This mesophilic bacterium HN001 showed a very high potential for hydrogen by fermentation. The maximum rate of hydrogen was 150 mmol-h 2 L-culture -1 h -1. The result was obtained at 47C and ph6.0. The reason was that 47 C is the suitable temperature for growth for this bacterium and metabolic pathway changes to acetate and ethanol at above this temperature. The yield of hydrogen increased along with the increase of cultivation temperature and the decrease of cultural ph. The highest yield of hydrogen was 2.4 mol-h 2 mol-glucose -1, at 50 C and ph 5.5. This bacterium was made clear to be the most expectable bacterium for hydrogen by fermentation. References: [1] T.de Vrije and P.A.M.Classen Dark hydrogen fermentations in Bio-methane & Bio-hydrogen ed J.H.Reith et al. (2003) 103123 ISBN: 9090171657 [2] Samir Kumar Khanal et al. Biological hydrogen : effects of ph and intermediate products. Int J Hydrogen Energy 29 (2004) 1123-1131 [3] Shigeharu Tanisho 2004. The Reason for Bacterial Evolution of Hydrogen by Fermentation and the Problems to be Solved for Utilization on Energy Production. The Hydrogen Energy Systems Society of Japan. 29 (1), 26 5/5